According to the CDC, greater than 1.1 million people in the United States and more than 35 million people worldwide are infected with HIV-1. While the introduction of combined antiretroviral therapy, cART, has greatly improved survival rates among AIDS patients, a substantial portion of HIV-1 infected individuals remain at risk for the development of full blown AIDS as a result of reactivation of latently infected cells, partly due t nonadherence to medication and emergence of drug resistant viruses. Moreover, HIV-1 positive long term survivors continue to develop comorbidities including an accelerated aging process, neurocognitive disorders, heart failure, and others. From the virological point of view, as none of the current treatments suppress viral gene transcription, it is suspected that low, yet continuous, levels of viral early proteins with regulatory and pathogenic activities may contribute to the development of these quality of life threating illnesses. Sadly, none of the efforts toward the development of vaccines against HIV-1 have shown promising outcomes. Thus, curing of AIDS by eradicating the HIV-1 genome in infected subjects requires a novel strategy that is specific, highly effective, sustained, and irreversible. Recently, we have adapted a genetic approach using the clustered regulatory interspaced short palindromic repeat-assisted system (Cas) and a short complementary single-stranded RNA, called guide RNA or gRNA, which specifically targets the U3 region of the HIV-1 LTR promoter and precisely excises a segment of the viral regulatory sequence required for its expression. In addition, the employment of single and multiplex gRNA in our Cas system show promising results that include eradication of the entire HIV-1 genome in latently infected microglial cells, thus abrogating viral gene expression and transcription. Based on this preliminary observation, we propose to develop an RNA-guided Cas9 that acts as molecular scissors and, by disrupting various regions of the LTR and/or removing the entire viral genome, abrogates reactivation of the virus in macrophages, microglia and astrocytes which serve as the viral reservoir in the brain. Furthermore, we will explore the feasibility of our single and multiplex Cas9 system for use as a prophylactic compound in in vitro HIV-1 infection culture models. The outcome of this molecular genetic and virological approach will provide a solid platform for developing preclinical and clinical studies toward the treatment f AIDS and its associated neurological and neurobehavioral disorders.